ASTM E3323-22

Standard Test Method for Lipid Quantitation in Liposomal Formulations Using High Performance Liquid Chromatography (HPLC) with an Evaporative Light-Scattering Detector (ELSD)

SIGNIFICANCE AND USE
5.1 Lipid composition in a liposomal formulation is an important aspect during synthesis of liposomes, which determines stability, surface characteristics, drug encapsulation, and drug release capabilities. The cholesterol component plays a key role in controlled drug release by adding stability to the liposome. A small variation in the lipid composition can significantly alter the parameters mentioned above (15).  
5.2 Variation in the lipid composition in the liposomal formulation may influence the safety and efficacy of the product. Therefore, chemical composition of the liposomes shall be determined.  
5.3 The pharmaceutical industry and regulatory agencies require QC, QA, specifications, thorough characterization, and quantification of lipid components (16, 17).  
5.4 This test method can be used to ascertain variations in the lipid component profiling of various liposomal formulations. However, this test method does not intend to identify chemical degradation products (18).  
5.5 Analyzing the stability of analytes and their chemical degradation profiles as a result of oxidation or hydrolysis is beyond the scope of this test method (18, 19).
SCOPE
1.1 This test method describes an analytical technique to quantify lipid components that are often present in liposomal formulations as major components.  
1.2 This test method uses high performance liquid chromatography (HPLC) to separate lipids in liposomal formulations and evaporative light-scattering detection (ELSD) to quantify the individual components.  
1.3 This test method quantifies three major organic components in liposomal formulations: cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000), and hydrogenated soy L-α-phosphatidylcholine (HSPC).  
1.4 This test method can estimate the absolute concentration of cholesterol, DSPE-PEG 2000, and HSPC and their ratio (DSPE-PEG 2000: HSPC: cholesterol) in liposomal formulations.  
1.5 This test method describes preparation of calibration standards and samples, HPLC and ELSD instrumentation, method development and method validation, sample analysis, and data reporting.  
1.6 The detection limits and quantitation limits for the analytes (lipid components) in this test method are in the range of 2 µg/g to 4 µg/g and 7 µg/g to 10 μg/g, respectively. The analytical measurement ranges for cholesterol, DSPE-PEG 2000, and HSPC are 10 µg/g to 165 µg/g, 10 µg/g to 300 µg/g, and 10 µg/g to 200 µg/g, respectively.  
1.7 Significant digits and rounding of all reported values have been performed according to the guidelines as established in Practice D6026.  
1.8 Units—The values stated in SI units are to be regarded as the standard. Where appropriate, c.g.s units in addition to SI units are included in this standard.  
1.9 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.10 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 30-Sep-2022
• ICS: 07.120 - Nanotechnologies
• Technical Committee: E56 - Nanotechnology
• Drafting Committee: E56.08 - Nano-Enabled Medical Products

Relations

• Refers: ASTM E3080-23 - Standard Practice for Regression Analysis with a Single Predictor Variable
• Effective Date: 01-Nov-2023
• Refers: ASTM E456-13a(2022)e1 - Standard Terminology Relating to Quality and Statistics
• Effective Date: 01-Apr-2022
• Refers: ASTM D1356-20a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Sep-2020
• Refers: ASTM D1356-20 - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 15-Mar-2020
• Refers: ASTM E3080-19 - Standard Practice for Regression Analysis with a Single Predictor Variable
• Effective Date: 01-Sep-2019
• Refers: ASTM E682-92(2019) - Standard Practice for Liquid Chromatography Terms and Relationships
• Effective Date: 01-Sep-2019
• Refers: ASTM E3080-17 - Standard Practice for Regression Analysis
• Effective Date: 01-Nov-2017
• Refers: ASTM E456-13A(2017)e3 - Standard Terminology Relating to Quality and Statistics
• Effective Date: 01-Oct-2017
• Refers: ASTM E456-13A(2017)e1 - Standard Terminology Relating to Quality and Statistics
• Effective Date: 01-Oct-2017
• Refers: ASTM E3080-16 - Standard Practice for Regression Analysis
• Effective Date: 01-Nov-2016
• Refers: ASTM D1356-15a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 15-Oct-2015
• Refers: ASTM D1356-15 - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Jul-2015
• Refers: ASTM D1356-14b - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Dec-2014
• Refers: ASTM E177-14 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-May-2014
• Refers: ASTM D1356-14a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-May-2014

Overview

ASTM E3323-22 is a standard test method developed by ASTM International for the quantitation of lipid components in liposomal formulations using high performance liquid chromatography (HPLC) coupled with an evaporative light-scattering detector (ELSD). The document offers a consistent and validated approach for measuring key lipid components essential for the stability, efficacy, and quality assurance of liposomal drug products.

Precise determination of lipids such as cholesterol, DSPE-PEG 2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), and HSPC (hydrogenated soy L-α-phosphatidylcholine) is critical in the pharmaceutical industry, supporting stringent regulatory requirements for quality control (QC), quality assurance (QA), and product characterization.

Key Topics

• Lipid Composition in Liposomal Formulations

  • Accurate quantitation of major organic lipid components is vital for drug delivery efficacy, liposome stability, and controlled drug release.
  • Variation in lipid composition influences product safety, efficacy, and regulatory compliance.

• Analytical Methodology

  • Utilizes HPLC for the separation of lipid components based on their chemical properties.
  • Employs ELSD for quantification, which is especially useful for analytes that do not have significant UV absorbance, making it suitable for a broad range of liposomal lipids.

• Calibration and Validation

  • Preparation of calibration standards and test samples
  • Method validation, including determination of detection and quantitation limits
  • Data reporting and significant digits management as per ASTM Practice D6026

• Scope and Limitations

  • The method ensures accurate profiling of cholesterol, DSPE-PEG 2000, and HSPC in liposomal formulations and their quantitative ratios.
  • Does not identify or quantify chemical degradation products or analyte stability post-oxidation or hydrolysis.

Applications

ASTM E3323-22 is widely applicable in the following areas:

• Pharmaceutical Development and Manufacturing

  • Routine QC and QA of liposomal drug products to ensure consistency in lipid composition
  • Supports compliance with regulatory agencies by fulfilling requirements for product characterization and batch release

• Research and Development

  • Characterization of new liposomal formulations and assessment of their stability profile
  • Optimization of lipid mixtures to enhance therapeutic delivery and product shelf life

• Regulatory Submissions

  • Provides robust, standardized data for submissions to regulatory bodies regarding the composition of lipid-based drug delivery systems

• Comparative Analysis

  • Assists in the assessment of manufacturing process variations and their impact on product quality

Related Standards

For comprehensive and compliant lipid quantitation and analytical practices, the following ASTM standards are referenced or aligned with ASTM E3323-22:

• ASTM D1193 - Specification for Reagent Water
• ASTM D1356 - Terminology Relating to Sampling and Analysis of Atmospheres
• ASTM D6026 - Practice for Using Significant Digits and Data Records in Geotechnical Data
• ASTM E131 - Terminology Relating to Molecular Spectroscopy
• ASTM E177 - Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• ASTM E682 - Practice for Liquid Chromatography Terms and Relationships
• ASTM E2490 - Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by Photon Correlation Spectroscopy
• ASTM E3025 - Guide for Tiered Approach to Detection and Characterization of Silver Nanomaterials in Textiles
• ASTM E3080 - Practice for Regression Analysis with a Single Predictor Variable

Practical Value

By establishing a consistent, validated, and internationally recognized method for lipid quantitation using HPLC-ELSD, ASTM E3323-22 supports:

• Improved product quality and process control in pharmaceutical manufacturing
• Enhanced regulatory compliance for liposomal drug products
• Reliable and reproducible quantitation of critical liposomal lipids
• Standardization across laboratories and organizations in both research and industry settings

ASTM E3323-22 serves as a key standard for laboratories and manufacturers focused on the development, regulation, and quality assurance of liposomal pharmaceutical formulations.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3323 − 22
Standard Test Method for
Lipid Quantitation in Liposomal Formulations Using High
Performance Liquid Chromatography (HPLC) with an
Evaporative Light-Scattering Detector (ELSD)
This standard is issued under the fixed designation E3323; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.9 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.1 This test method describes an analytical technique to
responsibility of the user of this standard to establish appro-
quantify lipid components that are often present in liposomal
priate safety, health, and environmental practices and deter-
formulations as major components.
mine the applicability of regulatory limitations prior to use.
1.2 This test method uses high performance liquid chroma-
1.10 This international standard was developed in accor-
tography (HPLC) to separate lipids in liposomal formulations
dance with internationally recognized principles on standard-
and evaporative light-scattering detection (ELSD) to quantify
ization established in the Decision on Principles for the
the individual components.
Development of International Standards, Guides and Recom-
1.3 This test method quantifies three major organic compo-
mendations issued by the World Trade Organization Technical
nentsinliposomalformulations:cholesterol,1,2-distearoyl-sn-
Barriers to Trade (TBT) Committee.
glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (DSPE-PEG 2000), and hydrogenated soy L-α-
2. Referenced Documents
phosphatidylcholine (HSPC).
2.1 ASTM Standards:
1.4 Thistestmethodcanestimatetheabsoluteconcentration
D1193Specification for Reagent Water
of cholesterol, DSPE-PEG 2000, and HSPC and their ratio
D1356Terminology Relating to Sampling and Analysis of
(DSPE-PEG 2000: HSPC: cholesterol) in liposomal formula-
Atmospheres
tions.
D6026Practice for Using Significant Digits and Data Re-
1.5 This test method describes preparation of calibration
cords in Geotechnical Data
standards and samples, HPLC and ELSD instrumentation,
D7439Test Method for Determination of Elements in Air-
method development and method validation, sample analysis,
borne Particulate Matter by Inductively Coupled Plasma-
and data reporting.
–Mass Spectrometry
E131Terminology Relating to Molecular Spectroscopy
1.6 The detection limits and quantitation limits for the
E177Practice for Use of the Terms Precision and Bias in
analytes(lipidcomponents)inthistestmethodareintherange
ASTM Test Methods
of 2µg⁄g to 4 µg/g and 7µg⁄g to 10 µg/g, respectively. The
E456Terminology Relating to Quality and Statistics
analytical measurement ranges for cholesterol, DSPE-PEG
E682Practice for Liquid Chromatography Terms and Rela-
2000, and HSPC are 10µg⁄g to 165 µg/g, 10µg⁄g to 300 µg/g,
tionships
and 10µg⁄g to 200 µg/g, respectively.
E2490Guide for Measurement of Particle Size Distribution
1.7 Significant digits and rounding of all reported values
of Nanomaterials in Suspension by Photon Correlation
havebeenperformedaccordingtotheguidelinesasestablished
Spectroscopy (PCS)
in Practice D6026.
E3025Guide for TieredApproach to Detection and Charac-
1.8 Units—The values stated in SI units are to be regarded
terization of Silver Nanomaterials in Textiles
asthestandard.Whereappropriate,c.g.sunitsinadditiontoSI
E3080Practice for Regression Analysis with a Single Pre-
units are included in this standard.
dictor Variable
This test method is under the jurisdiction of ASTM Committee E56 on
Nanotechnology and is the direct responsibility of Subcommittee E56.08 on
Nano-Enabled Medical Products. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Oct. 1, 2022. Published November 2022. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
approved in 2021. Last previous edition approved in 2021 as E3323–21. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/E3323-22. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3323 − 22
t
3. Terminology
A~t! 5 * ~S!dt (1)
t
3.1 Definitions:
Where A(t) and S(t) = Peak area and the instantaneous
3.1.1 accuracy, n—closeness of agreement between a test
detector’s response at time, t, respectively (2).
result and an accepted reference value.
3.1.11 coeffıcient of determination, n—statisticalmeasureof
3.1.1.1 Discussion—The term accuracy, when applied to a
the linear relationship between X and Y calculated by:
set of results, involves a combination of random components
n 2
and a common systematic error or bias component. E177
X Y
S D
( i i
3.1.2 aerosol, n—suspension of solid particles or liquid
i51
r 5 (2)
n n
droplets or both in a gaseous medium. D1356
X Y
S DS D
( i ( i
i51 i51
3.1.3 analyte, n—chemical constituent of interest in an
analytical procedure. E3025
Where n = number of observations. E131
3.1.4 analytical instrument qualification, n—collection of
3.1.12 evaporation, n—process by which an element or
documented evidence that an instrument performs suitably for
compound transitions from its liquid state to its gaseous state.
its intended purpose (1).
3.1.12.1 Discussion—In the ELSD technique, only the sol-
vent and volatile buffer components of the HPLC-effluent are
3.1.5 baseline noise, n—combinationofhigh-frequencysig-
removed during the evaporation process and less-volatile or
nal fluctuations and low-frequency signal drift that affect
nonvolatile analytes are left behind as dried particles. This
baseline stability.
process is also called “desolvation”. The evaporation process
3.1.5.1 Discussion—These signal fluctuations can originate
depends on various factors including gas pressure, flow rate of
from line-voltage fluctuations, shot noise (Poisson noise) from
the carrier gas, nature of the solvents, and temperature of the
electronic circuits, improper solvent degassing, temperature
evaporation tube. It is important to choose the appropriate
instability,andothernonequilibriumeffects.Noiseisrepresen-
mobile phase components that are volatile; nonvolatile buffers
tative of detector response that is not related to responses from
are not compatible with this test method.
analytes or matrix interferences.
3.1.13 intermediate precision, n—closeness of agreement
3.1.6 calibration curve, n—relationship between measured
between test results obtained under specified intermediate
response values and analytical concentrations of a standard or
precision conditions. E177
reference material. D7439
3.1.6.1 Discussion—A set of calibration standards are used
3.1.14 intermediate precision conditions, n—conditions un-
to construct a calibration curve, and the concentration of
der which test results are obtained with the same test method
analyte present in an unknown sample can be determined by
using test units taken at random from a single quantity of
comparing the detector response with the calibration curve.
material that is as nearly homogeneous as possible and with
changing conditions such as operator, measuring equipment,
3.1.7 calibration standards, n—set of solutions with known
location within the laboratory, and time. E177
analyte concentration used to construct calibration curves.
3.1.15 limit of detection, n—least amount of analyte in a
3.1.8 carryover effect, n—systematic error that is derived
sample that can be detected but not necessarily quantitated
from the preceding sample injection being introduced into the
under the stated experimental conditions.
next sample affecting accurate quantitation.
3.1.15.1 Discussion—The limit of detection is usually ex-
3.1.9 cholesterol, n—steroidal organic compound that stabi-
pressed as the concentration of the analyte in the test sample.
lizes the lipid bilayer in liposomal formulations.
3.1.16 limit of quantitation, n—least amount of analyte in a
3.1.10 chromatogram, n—graphical presentation of detector
sample that can be quantitatively determined with suitable
response plotted as a function of elution time or effluent
precision and accuracy.
volume as the sample components elute from the column and
3.1.16.1 Discussion—The limit of quantitation is usually
reach the detector.
expressedastheconcentrationoftheanalyteinthetestsample.
3.1.10.1 Discussion—In the case of evaporative light-
3.1.17 linearity, n—ability of the analytical method (within
scattering detection (ELSD), the detector response is often
a certain range) to obtain test results that are directly propor-
expressed as voltage (mV) over a range of elution time (t),
tional to the concentration (amount) of the analyte in the
wherevoltageisafunctionoftheintensityoflightscatteredby
sample (3).
nonvolatile particles inside the optical chamber. For analysis,
3.1.17.1 Discussion—To establish response linearity, a
the characteristic response of ELSD for an eluting analyte is
minimum of six analyte concentrations are recommended.
typically evaluated from the peak area under the curve that is
Regression analysis by the method of least squares (r )
recorded in the chromatogram. This peak area (A) can be
provides a mathematical estimate of the degree of linearity.
expressed mathematically as an integral of detector response
for analytes over an elution time interval from t to t :
3.1.18 lipids, n—diverse group of organic compounds that
1 2
are soluble in organic solvents but are insoluble in water.
3.1.18.1 Discussion—In this test method, lipids refer to
cholesterol, DSPE-PEG 2000, and HSPC. The chemical struc-
The boldface numbers in parentheses refer to a list of references at the end of
this standard. tures of these three lipids are presented in Appendix X1.
E3323 − 22
3.1.19 liposomal formulation, n—product designed to assist 3.1.30 regression analysis, n—statistical procedure used to
in the delivery of an active pharmaceutical ingredient, either characterize the association between two or more numerical
encapsulated or intercalated in a liposome. variables for prediction of the response variable from the
3.1.19.1 Discussion—Formulated products can contain predictor variable. E3080
vesicles having a single lipid bilayer (unilamellar), multiple
3.1.30.1 Discussion—Theobjectiveistoobtainaregression
concentric lipid bilayers (multilamellar), or a mixture of model for use in predicting the value of the response variable
unilamellar and multilamellar vesicles. for given values of the predictor variable. In this test method,
the response variable is the ELSD signal [light-scattering unit
3.1.20 liposome, n—synthetic vesicle composed of a one or
(LSU)] and the predictor variable is mass concentration.
more bilayers formed by amphipathic molecules such as
phospholipids that enclose a central aqueous compartment. 3.1.31 repeatability, n—precision of test results from tests
Adapted from (4). conductedwithintheshortestpracticaltimeperiodonidentical
materialbythesametestmethodinasinglelaboratorywithall
3.1.21 matrix blank, n—substance that closely matches the
known sources of variable conditions controlled at the same
samples being analyzed with regard to matrix components.
levels. Adapted from E177
3.1.21.1 Discussion—Ideally, the matrix blank contains all
the sample components except the analyte(s) of interest and is 3.1.32 reproducibility, n—precisionoftestresultsfromtests
conducted on identical material by the same test method in
subjected to all sample-processing operations including all
reagents used to analyze the test samples. The matrix blank is different laboratories. Adapted from E456
used to determine the presence or absence of any significant
3.1.33 robustness, n—measure of change in the outcome of
interference as a result of the matrix, reagents, and equipment.
an analytical procedure with deliberate and systematic varia-
3.1.22 matrix effect, n—influence of one or more compo- tions in any or all of the key method parameters that influence
nents from the sample matrix on the measurement of the it. Adapted from E2490
analyte concentration or mass.
3.1.34 solvent blank, n—solution containing all reagents
3.1.22.1 Discussion—Matrix effects may be observed as
used in sample dissolution in the same quantities used for
increasedordecreaseddetectorresponsescomparedwiththose
preparation of blank and sample solutions.
produced by simple solvent solutions of the analyte (5).
3.1.34.1 Discussion—The solvent blank is used to assess
3.1.23 method validation, n—process used to confirm that
contamination from the laboratory environment and character-
ananalyticalprocedureusedforaspecifictestissuitableforits
ize spectral background from the reagents used in sample
intended purpose.
preparation. D7439
3.1.24 mobile phase, n—liquid used to elute sample com-
3.1.35 specificity, n—ability to assess unequivocally the
ponents through the column that may consist of a single
analyte in the presence of components that may be expected to
component or a mixture of components.
be present in the test sample.
3.1.24.1 Discussion—The term eluent is often used for the
3.1.35.1 Discussion—Typically, these might include
preferred mobile phase. E682
impurities, degradants, matrix, and so forth (3).
3.1.25 nebulization, n—process to convert the solution of
3.1.36 system suitability, n—determination of instrument
target analyte components to a fine-spray via a nebulizer.
performance in a particular procedure (for example, sensitivity
3.1.26 peak area, n—area under a peak obtained from and chromatographic retention) by analyzing a set of appro-
integration of a detector signal above the baseline for a given priate reference standards before the analytical run.
component.
3.2 Definitions of Terms Specific to This Standard:
3.1.27 peak resolution, n—measure of chromatographic
3.2.1 test sample, n—final form of the sample that is used
separation of two components in a mixture calculated by:
for testing.
3.2.1.1 Discussion—In this test method, the sample solubi-
t 2 t
~ !
R2 R1
R 52 3 (3)
s
lized in methanol followed by appropriate dilution by solvent
w 1 w
~ !
1 2
is defined as the test sample.
where:
3.2.2 test unit, n—unit or portion of a material that is
R = peak resolution,
s
obtained from a primary material following a sampling proce-
t and t = retention time of the two components 1 and 2
R2 R1
dure to acquire test result(s) for the property(-ies) to be
(t > t ), and
R2 R1
measured.
w and w = corresponding widths at the bases of the peaks
1 2
3.2.2.1 Discussion—In this test method, the original lipo-
obtained by extrapolating the relatively
somal formulation to be tested for lipid quantitation is defined
straight sides of the peaks to the baseline.
as the test unit.
3.1.28 precision, n—closeness of agreement between inde-
3.3 Acronyms:
pendent test results obtained under stipulated conditions. E177
3.3.1 Cal—Calibration
3.1.29 range, n—interval between the upper and lower
3.3.2 CRM—Certified reference material
concentrations of the analyte in a sample for which it has been
demonstrated that the analytical procedure has an acceptable 3.3.3 DSPE-PEG—1,2-Distearoyl-sn-glycero-3-phospho-
level of accuracy, precision, and linearity. ethanolamine-N-methoxy(polyethylene glycol)
E3323 − 22
3.3.4 ELSD—Evaporative light-scattering detector regression analysis are used to quantify the individual mass
(concentration) of lipid components in an unknown liposome
3.3.5 HPLC—High performance liquid chromatography
sample.
3.3.6 HSPC—Hydrogenated soy L-α-phosphatidylcholine
4.5 This test method describes the specific test conditions,
3.3.7 ID—Inside diameter
sample preparation, method validation, and data analysis
3.3.8 LOD—Limit of detection
requirements.
3.3.9 LOQ—Limit of quantitation
5. Significance and Use
3.3.10 LSU—Light-scattering unit
5.1 Lipid composition in a liposomal formulation is an
3.3.11 OSHA—Occupational Safety and HealthAdministra-
important aspect during synthesis of liposomes, which deter-
tion
minesstability,surfacecharacteristics,drugencapsulation,and
3.3.12 QA—Quality assurance
drug release capabilities. The cholesterol component plays a
key role in controlled drug release by adding stability to the
3.3.13 QC—Quality control
liposome. A small variation in the lipid composition can
3.3.14 RCF—Relative centrifugal force
significantly alter the parameters mentioned above (15).
3.3.15 RSD—Relative standard deviation
5.2 Variation in the lipid composition in the liposomal
3.3.16 SD—Standard deviation
formulation may influence the safety and efficacy of the
3.3.17 SLM—Standard liter per minute product. Therefore, chemical composition of the liposomes
shall be determined.
3.3.18 ULOQ—Upper limit of quantitation
5.3 The pharmaceutical industry and regulatory agencies
3.3.19 UV—Ultraviolet
require QC, QA, specifications, thorough characterization, and
quantification of lipid components (16, 17).
4. Summary of Test Method
5.4 This test method can be used to ascertain variations in
4.1 Over the past few decades, several liposomal drug
the lipid component profiling of various liposomal formula-
formulations have been approved for clinical use (6, 7).An
tions. However, this test method does not intend to identify
ongoing effort from pharmaceutical industries, academic
chemical degradation products (18).
institutions, foundations, and industry partners has been made
to develop new nanoscale liposomal formulations with im-
5.5 Analyzing the stability of analytes and their chemical
proved drug efficacy, and many products are currently being
degradation profiles as a result of oxidation or hydrolysis is
assessed in clinical trials (7-11). The critical quality attributes
beyond the scope of this test method (18, 19).
to consider for these nanomaterials include size and shape
heterogeneity, chemical composition, and physicochemical
6. Interferences
stability of ingredients present in the liposome formulation
6.1 Method interferences may be introduced by impurities
(12).
presentinreagents,glassware,andotherapparatususedduring
4.2 This test method describes an analytical method for the
samplepreparationandinstrumentalanalysis.Theseimpurities
separation and quantitation of cholesterol, DSPE-PEG 2000,
may result in high baseline noise or interfering peaks. The
and HSPC in liposomal formulations.
presence and magnitude of method interferences are deter-
4.3 This test method is based on the combination of two mined by routine analysis of solvent and laboratory blanks.
well-established analytical techniques: (1) chromatographic
6.2 ToavoidheterogeneityinpHofthesolution,eluentsand
separation of analytes (a mixture of cholesterol, DSPE-PEG
buffer salts shall be properly mixed (sonication is recom-
2000, and HSPC) via HPLC using a designated column, and
mended). It is also recommended that the solvent reservoir
(2) quantitation of analytes present in the effluent via ELSD in
bottlebecleanedroutinely,andtheappropriatebottlesbefilled
which the scattering intensity of the incident light is correlated
with freshly prepared mobile phase solutions.
with the total mass of the analytes present in the injected
6.3 Aerosol formation in ELSD requires a constant supply
sample. The ELSD detector can serve as a universal detector
for a range of nonvolatile or semi-volatile analytes including of dry and filtered gas that is free from particulate matter and
nonvolatile hydrocarbons. The most commonly used gas is
those that do not have ultraviolet (UV)-absorbing chro-
mophores (13, 14). nitrogen.Theinletgasshallbefilteredthrougha0.01µmfilter
to remove particulate matter, and an appropriate gas adsorbent
4.4 A calibration curve from six calibration standards of
trap should be used to remove nonvolatile hydrocarbons and
cholesterol, DSPE-PEG 2000, and HSPC is developed by
moisture, and thereby minimizing baseline noise. The use of
following the procedure described in this test method. As
gases that allows either combustion of solvents or oxidation of
ELSD shows a nonlinear response with analyte concentration,
target analytes should be avoided.
logarithmic transformation is performed to obtain a linear
regression model. Hence, log (peak area) versus log (concen- 6.4 Keeptheautosamplerinjectionportandcolumncleanto
tration) is plotted for each analyte to obtain the corresponding avoid carryover or ghost peaks. Contamination of glass con-
calibration curve.The slope and intercept obtained from linear tainers or vials used for this test method should be avoided.
E3323 − 22
6.5 Fluctuations in detector response can adversely affect 7.11 Ultrasonic water bath.
data quality. To avoid this issue, stabilize the detector at the
8. Reagents and Materials
experimental conditions (for example, nebulization
temperature, evaporation temperature, and nitrogen flow rate)
8.1 Purity of Reagents—Reagent-grade chemicals shall be
for 30 min. This will also minimize baseline noise.
used in all tests. Unless otherwise indicated, it is intended that
all reagents conform to the specifications of the Committee on
6.6 Set the temperature of the column compartment as
Analytical Reagents of theAmerican Chemical Society where
recommended in the test method and equilibrate the column
such specifications are available. Other grades may be used,
while stabilizing the detector response (see 6.5).
provided it is first ascertained that the reagents are of suffi-
6.7 The presence of bubbles in the HPLC tubing causes
ciently high purity to permit their use without lessening the
pressure fluctuations. Solvent purging (2.00 mL/min) for
accuracy of the determinations.
15min in which the column is bypassed is recommended to
8.2 Reagents—All reagents should be of LC-MS grade and
remove interferences caused by the presence of air bubbles.
solvents should be prefiltered with a ≤0.2µm filter. Other
6.8 Excipientspresentinthetestsamplesmayinterferewith
grades (for example, HPLC grade) may be used, provided it is
the detection of cholesterol, DSPE-PEG 2000, or HSPC.
first ascertained that the reagents are of sufficiently high purity
Therefore, the matrix effect can be assessed by comparing the
to permit their use without lessening the accuracy of the
detector response for a known amount of standard analyte
determinations.
spiked in the matrix blank and for the same amount of analyte
8.2.1 Ammonium acetate, LC-MS grade.
spiked in a solvent blank. If any matrix effect is observed by
8.2.2 Acetonitrile, LC-MS grade.
this comparison, optimization of the sample preparation pro-
8.2.3 Methanol, LC-MS grade.
cedure to remove the interfering compounds from the test
8.2.4 Purity of Water—Unless otherwise indicated, refer-
sampleormodificationofthechromatographicparameters(for
ences to water shall be understood to mean reagent water as
example, solvent gradient or eluent flow rate) to avoid the
definedbyType1ofSpecificationD1193.Usedeionizedwater
coelution of target analytes and interfering excipients are
(>18 MΩcm) Type 1 high-purity water.
recommended. The Bligh-Dyer method, as described in Ap-
8.3 Materials:
pendix X3, could be adopted to remove water-soluble excipi-
8.3.1 Lipid Standards:
ents in test liposomal formulations, if necessary.
8.3.1.1 Cholesterol, ≥99 % pure, powder form.
6.9 Chemicals with high purity shall be used for the
8.3.1.2 HSPC, ≥99 % pure, consisting of C16:0 (HSPC 1)
preparation of lipid calibration standards. When feasible, it is
and C18:0 (HSPC 2) fatty acids, powder form.
recommended that higher-order reference standards (for
8.3.1.3 DSPE-PEG 2000, ≥99 % pure, powder form.
example, CRMs) are used in calibration. If reference materials
arenotavailable,high-qualitycrystallineorlyophilizedchemi-
9. Hazards
cals of known purity can be used for this purpose.
9.1 BecausetheELSDproducesanaerosol,theoutletofthe
6.10 All the stock solutions, calibration standards (calibra-
ELSD unit shall be vented to a suitable fume hood or external
tion levels), and test samples should be stored either at 0°C to
exhaust.
4 °C or –20°C as recommended in this test method to avoid
9.2 Follow laboratory safety protocol and proper protective
degradation of target analytes.
measures while handling liposomal formulations.
7. Apparatus
9.3 This test method uses methanol, chloroform, and ac-
7.1 HPLC, with in-line degasser module.
etonitrile that exhibit various levels of toxicity through inha-
lation and skin contact. All organic solvents used in this test
7.2 ELSD.
method should be handled in a chemical fume hood. Avoid
7.3 Deactivated BEH (bridged ethylene hybrid) C18
inhalation of the solvents. All the organic wastes should be
column, with 13 nm pore size, 3.5 µm particle size, and 3 mm
disposed of appropriately.
ID × 150 mm column length or other equivalent stable C18
9.4 The waste of the sample extract should be handled with
column that can resolve the peaks for analytes and potential
proper care and precaution. Follow laboratory safety protocol
interference with a peak resolution ≥1.5.
to appropriately dispose of used test sample. The user is
7.4 Analytical balance that can accurately weigh with
advised to follow relevant local regulatory requirements (for
≤0.0001g readability.
example, OSHA), suppliers’ safety data sheets, institutional
7.5 Vortex mixer. requirements, and recommended procedures pertaining to safe
handlinganddisposalofallchemicalsusedinthistestmethod.
7.6 Mechanical pipettors, covering ranges from 2µL to
20µL, 100µL to 200µL, and 100µL to 1000 µL and 10 mL.
7.7 Glass amber vials, 10mL and 20 mL.
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
Standard-Grade Reference Materials, American Chemical Society, Washington,
7.8 Autosampler amber vials, 2 mL.
DC. For suggestions on the testing of reagents not listed by theAmerican Chemical
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
7.9 Solvent reservoir bottle,1L.
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
7.10 Bottle top vacuum filter system, pore size 0.2 µm. copeial Convention, Inc. (USPC), Rockville, MD.
E3323 − 22
containersusingasecondarycontainerarehighlyrecommendedpractices.
10. Preparation of Mobile Phases
NOTE 4—The user is required to provide a certificate of analysis or
10.1 Mobile Phase A: Acetonitrile/Water (90/10 v/v) +
equivalent alternative information on the source, purity, storage
5 mmol ⁄L Ammonium Acetate:
conditions, retest/expiration date, and lot/batch number of reference
standards or high-purity chemicals to ensure quality and stability (20).
10.1.1 Filter deionized water (>18 MΩ cm) through a
0.2µmmembraneusingabottletopvacuumfiltersystem.This
11.1 Powder stocks of the analytes stored in the freezer at
step is not needed in the case of a water purification system
–20ºCshouldbeallowedtoequilibrateatambienttemperature
with an attached 0.2 µm filter.
before weighing.
10.1.2 Rinse an empty solvent bottle (1 L) with deionized
11.2 Deionized water (>18 MΩ cm) should be filtered
water thoroughly and dry the bottle.
through a 0.2 µm membrane using a bottle top vacuum filter
10.1.3 Ina1Lvolumetric flask, transfer 3.85g 6 0.02 g of
system before use. This step is not needed in the case of water
ammonium acetate quantitatively, dissolve the salt thoroughly
purification system with an attached 0.2 µm filter.
with ≈800 mL of deionized water, equilibrate the solution at
room temperature, and then fill the flask with deionized water 11.3 Preparation of Individual Stock Solutions—Prepare
individual stock solutions (≈1000 µg/g) of cholesterol, DSPE-
to the 1 L graduation mark. This will provide 50mmol⁄L
solution of ammonium acetate. PEG 2000, and HSPC in separate amber glass vials (20 mL).
10.1.4 Transfer 100 mL of 50 mmol/L ammonium acetate 11.3.1 Rinse three empty 20 mL amber glass vials with
and 900 mL of LC-MS-grade acetonitrile in the clean and dry deionized water and dry thoroughly.
1 L bottle. The volumes are measured using a graduated 11.3.2 Label each vial with the corresponding analyte j,
cylinder (100 mL and 1 L, respectively). where j = cholesterol, DSPE-PEG 2000, and HSPC. Put a cap
10.1.5 Degas the solution for 10min to 15 min using an on each bottle.
ultrasonicbathbeforeusingitasmobilephaseA.Theuseofan
11.3.3 Weigh each capped vial on an analytical balance and
in-line degasser module further helps to achieve a stable record the mass to 60.1 mg as W .
0j
baseline during an analytical run.
11.3.4 Individually weigh 10mg 6 2 mg of cholesterol,
10.1.6 Transfer the degassed solution (mobile Phase A) to
DSPE-PEG 2000, and HSPC on an analytical balance, transfer
an empty, clean, and dry 1 L reservoir attached to the HPLC.
eachanalytetotheappropriatelylabeledvial,putacaponeach
vial, and record the mass of the capped vial + analyte as W .
1j
10.2 Mobile Phase B: Methanol + 5 mmol/L Ammonium
11.3.5 Transfer 12.5 mL (≈10.0 g) of LC-MS-grade metha-
Acetate:
nol using a mechanical pipettor to the vial from 11.3.2 labeled
10.2.1 To prepare 1 L of mobile Phase B, weigh 385mg 6
as cholesterol. Put a cap on each vial.
10mgofammoniumacetateandtransferitquantitativelytoan
11.3.6 Repeat 11.3.5 for DSPE-PEG 2000 and HSPC.
empty 1 L volumetric flask, fill the flask with ≈800 mL of
11.3.7 Weigh each capped vial containing, analyte j, and
LC-MS-grade methanol to dissolve ammonium acetate, equili-
methanol on an analytical balance and record the mass to
brate the solution at room temperature, and then fill the flask
62mgas W .
with methanol to the 1 L graduation mark to make a homoge-
2j
11.3.8 Dissolve the solids in each vial thoroughly by vortex
neous 5 mmol/L ammonium acetate solution in methanol.
mixing for 1 min.
10.2.2 Transfer the methanolic 5 mmol/L ammonium ac-
etate solution to a clean and dry 1 L bottle.
NOTE 5—Complete solubilization and formation of a homogeneous
10.2.3 Sonicate the solution to homogenize the analytes as
solution may require an additional 5 min bath sonication.
needed and degas the solution for 10min to 15 min using an
11.3.9 Store the vials of the individual stock solutions at
ultrasonicbath.Useofanin-linedegassermodulefurtherhelps
–20°C until needed. Stock solutions are stable up to four
to achieve a stable baseline during an analytical run.
months under this condition.
10.2.4 Transfer the degassed methanol with 5 mmol/L
11.3.10 The individual stock concentrations of the three
ammoniumacetatetoanempty,clean,anddry1Lreservoiron
components, C,arecalculatedusingEq4.Thepurity(j)%for
j
HPLC.
each component should be the value from the manufacturer’s
NOTE 1—Sparging with helium can be used as an alternative to the
certificate of analysis:
ultrasonic degassing and in-line vacuum degassing combination as rec-
Massoftheanalyte
ommended in this test method.
C 5 5
j
Totalmassofsolution
11. Preparation of Calibration Standards purity j % 3 W 2 W ⁄ W 2 W 310 µg/g (4)
~ ~ ! ! @~ ! ~ !#
1j 0j 2j 0j
NOTE 2—Gravimetric Measurements—All working solutions in this
Where j = cholesterol, DSPE-PEG 2000, or HSPC.
test method are prepared gravimetrically using an analytical balance
(0.0001 g accuracy).The concentration of an analyte is expressed in units
11.4 Preparation of Individual Stock Solutions—Prepare
ofµganalytepergofsolution.Althoughthevolumetricpreparationshows
individual stock solutions (≈500 µg/g) of cholesterol, DSPE-
close agreement with the gravimetric preparation, it is known that a 1%
PEG 2000, and HSPC in separate amber glass vials (20 mL).
to 5 % error can be introduced during small volume transfers and, hence,
bias the quantitation results. The analytical balance provides better
11.4.1 Rinse three empty 20 mL glass vials with deionized
measurementresolution(thatis,moresignificantfigures)thanmechanical
water and dry thoroughly.
pipettes and offers better accuracy.
11.4.2 Label each vial with the corresponding component
NOTE 3—Conditioning the pipette tip with appropriate solvents before
(analyte) j, where j = cholesterol, DSPE-PEG 2000, or HSPC.
transfer of calibration standards for weighing, working promptly with the
stock solutions, and weighing the volatile liquids in securely capped Put a cap on each vial.
E3323 − 22
11.4.3 Follow 11.3.3 – 11.3.8 (with a lesser amount of solution and the total mass of analyte j plus solvent (that is,
analyte j, for example, 5mg 6 1 mg) to obtain Cj values of methanolic solution of analyte j).
500µg⁄g. 11.5.8 The concentrations of six stock solutions for three
11.4.4 The concentration of each analyte j in the individual analytes, C (µg/g) (≈1000 µg/g and ≈500 µg/g) are precalcu-
j
stock solutions is calculated using Eq 4. lated in 11.3.10 and 11.4.5, respectively.These values are used
11.4.5 Store the vials of the individual stock solutions at in 11.5.9 to calculate the concentration of a calibration stan-
–20°C until needed. Stock solutions stored under this condi- dard.
tion are stable up to four months. 11.5.9 Thefinalconcentrationofthecalibrationstandardfor
analyte j is determined by Eq 5:
11.5 Preparation of Calibration Standards for Individual
cal
C µg/g 5 C 3 A 2 A ⁄ A 2 W (5)
Analyte: ~ ! @~ ! ~ !#
j j 2j 1j 1j 0j
11.5.1 Six calibration standards (that is, calibration levels)
11.5.10 Repeat 11.5.2 – 11.5.9 for calibration levels 2 to 6,
of individual analytes: (1) cholesterol with target concentra-
using volumes of each analyte stock solution and methanol
tions ranging from 5µg⁄g to 175 µg/g (for example, 5µg⁄g,
shown in Table 1.
10µg⁄g,25µg⁄g,50µg⁄g,100µg⁄g,and175µg/g);(2)DSPE-
11.5.11 Repeat 11.5.2 – 11.5.10 for the other two analytes,
PEG 2000 with target concentrations ranging from 5µg⁄g to
j = DSPE-PEG 2000 and HSPC.
300 µg/g (for example, 5µg⁄g, 10µg⁄g, 50µg⁄g, 100µg⁄g,
11.5.12 Homogenize each individual analyte + methanol
150µg⁄g, and 300 µg/g); and (3) HSPC with target concentra-
solutionforcalibrationlevels1to6byvortexmixing.Storethe
tions ranging from 5µg⁄g to 200 µg/g (for example, 5µg⁄g,
calibrationsolutionsat0°Cto4°C(foroneday)or–20°Cfor
10µg⁄g, 50µg⁄g, 100µg⁄g, 150µg⁄g, and 200 µg/g) are
longer storage. The solutions are stable up to one month at
prepared from the 1000 µg/g and 500 µg/g stock solutions (see
–20°C.
11.3 and 11.4 respectively). ELSD has stronger analytical
11.5.13 A representative table for the six calibration stan-
sensitivity for cholesterol compared to DSPE-PEG 2000 and
dards with the target analyte concentrations is provided in
HSPC, therefore the highest calibration level for cholesterol is
Table 1.
targetedat ≈175µg/g.Inthistestmethod,allmeasurementsare
NOTE 6—The concentrations provided in Table 1 are the examples of
gravimetric, and therefore, the user does not need to transfer
target concentrations for various calibration levels. Eq 5 shall be used to
theexactvolumeofmethanoloranalytestocksolutiontoreach
obtain mass-based concentrations for any target analyte concentration.
the exact target analyte concentration but does need to record
11.5.14 The individual calibration plots are used only to
the accurate mass and calculate the concentration of each
verify the calibration range in which the regression model and
calibration standard by following the example described in
thefitremainslinear,andtochoosetheconcentrationrangefor
11.5.7 – 11.5.9.
preparing calibration standards containing mixture of the three
11.5.2 Rinse one empty 10 mL glass vial with deionized
analytes.
water and dry thoroughly.
11.5.3 Rinse one measuring cylinder with deionized water 11.6 Preparation of Calibration Standards of Analyte Mix-
and dry thoroughly. tures:
cal
11.5.4 For calibration Level 1, C = 5 µg/g, where j =
11.6.1 Mixturesofcholesterol,DSPE-PEG2000,andHSPC
j
cholesterol, DSPE-PEG 2000, or HSPC. Label the 10 mL vial are utilized to construct calibration curves containing a mini-
as Level 1 (cholesterol, DSPE-PEG 2000 or HSPC). Put a cap
mum of six calibration levels, and the constructed calibration
on the vial. curve within the bracketed range is used for method validation
11.5.5 For that level, weigh the capped vial on an analytical and the quantitation of all three analytes present in the test
balance and record the mass to 60.1 mg as W . samples.
0j
11.5.6 Addthevolumeofthestocksolutionwithanalyte j= 11.6.2 Each calibration standard (level) is obtained by
cholesterol to the vial as given in Table 1 for Level 1. Place a combining the three stock solutions of individual analytes
cap on the vial. Record the mass as A in units of g. (either ≈1000µg⁄g or ≈500 µg/g stock) in a 1:1:1 mass ratio
1j
11.5.7 Add the volume of methanol to the vial as given in with methanol in a 10mL vial. Example of solution prepara-
Table 1 for Level 1. Place a cap on the vial. Record the mass tion parameters for various concentration levels (mass ratio of
as A in units of g.The three masses, W , A , and A , will be cholesterol: DSPE-PEG 2000: HSPC ≈ 1:1:1) are summarized
2j 0j 1j 2j
required to calculate the actual mass of analyte j present in the in Table 2.
A
TABLE 1 Example of Six Calibration Standards for Individual Analyte Prepared from Two Stock Solutions (1000 and 500 µg/g)
Amount of analyte needed (C)
Target conc. of analyte Conc. of stock used Total mass of the j Amount of methanol
Cal. level
in methanol (µg/g) (ppm, µg/g) = C target cal. standard (g) added (mL)
j Mass (µg) Stock Volume (mL)
1 5 500 4 20 0.05 5.00
2 10 500 4 40 0.10 4.95
3 50 500 4 200 0.25 4.80
4 100 500 4 400 0.50 4.55
5 150 1000 4 600 0.76 4.29
6 300 1000 4 1200 1.52 3.35
A
To estimate the volume of analyte stock solution, the density of methanol is used as the density of stock. Other calibration standards with intermediate analyte
concentration, for example, concentrations of 25 µg ⁄g, 75 µg ⁄g, 175 µg ⁄g, or 200 µg/g can be prepared similarly.
E3323 − 22
A
TABLE 2 Example of Calibration Standards of Analyte Mixtures Prepared from Two Stock Solutions (1000 and 500 µg/g)
Target conc. of each
Amount of analyte needed (C)
j
Conc. of stock used Total mass of the Amount of methanol
Cal. level analyte in the mix
(ppm, µg/g) = C target cal. standard (g) added (mL)
j
Mass (µg) Stock Volume (µL)
(µg/g)
1 5 500 2 10 25 2.43
210 500 220 50 2.38
315 500 230 75 2.30
4 25 500 2 50 125 2.15
5 50 1000 2 100 125 2.15
6 75 1000 2 150 188 1.95
7 200 1000 2 400 500 1.00
8 300 1000 2 600 750 0.25
A
To estimate the volume of analyte stock solution, the density of methanol is used as the density of stock. The analyst is not limited to example concentrations, and any
sixconcentrations(evenlyspaced)canbeusedtoestablishalinearfitwithr $0.995.Othercalibrationstandardswithintermediateanalyteconcentration,canbeprepared
similarly.
11.6.3 Topreparetheeightlevelsofthecalibrationstandard thoroughly using vortex mixer. Masses in each step shall be
mixtures shown in Table 2, follow the steps similar to those recorded.The component concentrations in the QC sample are
described in 11.5.2 – 11.5.7. determined based on the masses of each of three analytes and
11.6.4 Rinse the eight empty 10 mL glass vials with the mass of methanol solvent that have been added.
deionized water and dry thoroughly.
12.3 To assess the specificity: (1) prepare a mixture of
11.6.5 Label each vial with the corresponding calibration
cholesterol, DSPE-PEG 2000, and HSPC having each analyte
level (1 to 8).
concentration of 50 µg/g in methanol as described in Table 2;
11.6.6 Add appropriate volumes of the three individual
(2) prepare a matrix blank which is a methanol solution
stock solutions (cholesterol, DSPE-PEG 2000, and HSPC) and
containing possible matrices that are expected to be present in
methanol as given in Table 2 for the calibration level. Place a
the test sample (that is, liposomal formulation); (3) prepare a
cap on each vial.
solvent blank containing only methanol; and (4) prepare three
11.6.7 Record the mass of each capped vial after the
independent solutions of cholesterol (50 µg/g), DSPE-PEG
addition of each analyte solution.
(50µg⁄g), and HSPC (50µg⁄g) in methanol as described in
11.6.8 Add the appropriate volume of methanol to each vial
Table 1.
asgiveninTable2forthecalibrationlevel.Placeacaponeach
12.4 For the accuracy study, prepare three replicates in
vial.
methanol of a set of samples with the following analyte
11.6.9 Record the mass of each capped vial after the
concentrations: (1) (low) mixture of cholesterol, DSPE-PEG
addition of methanol.
2000, and HSPC with each analyte concentration of 25 µg/g;
11.6.10 The final concentration of an individual analytes j
(2) (medium) mixture of cholesterol, DSPE-PEG 2000, and
(where j = cholesterol, DSPE-PEG 2000, or HSPC) in a
HSPC with each analyte concentration of 50 µg/g; and (3)
calibration standard mixture can be calculated as:
(high) mixture of cholesterol, DSPE-PEG 2000, and HSPC
cal
C µg/g 5
~ !
j
with each analyte concentration of 100 µg/g (see Table 2 for
massofanalyteadded, µg ⁄ totalmassofanalyte+methanol,g
@~ ! ~ !#
preparation parameters). The exact analyte concentrations for
(6)
each set are calculated from the measured masses of the
analyte(s) present in a measured amount of solution (analyte +
Where the mass of analyte j added (µg) = C × (mass of the
j
methanol).
individual stock solution added, g).
11.6.11 Homogenize the mixture by vortex mixing the
12.5 For repeatability (that is, method precision), the same
solution. Store these calibration standard mixtures at 0°C to set of solutions that have been prepared in 12.4 can be used.
4°C(foroneday)or–20°Cforlongerstorage.Stocksolutions
12.6 To assess matrix effects, prepare three samples in
are stable up to one month at –20°C.
methanolthatcontainalloftheexpectedmatrixcomponentsat
expectedconcentrationlevelsasinthetestsample.Theanalyte
12. Preparation of Samples for QC, Method Specificity,
concentrations chosen for this study were (low, medium, and
Recovery Experiment, and Method Precision
high): (1) cholesterol (25 µg/g), DSPE-PEG 2000 (25 µg/g),
12.1 QC samples are to be prepared by mixing cholesterol,
and HSPC (25 µg/g); (2) cholesterol (50 µg/g), DSPE-PEG
DSPE-PEG 2000, and HSPC from the 500 µg/g stock solu-
2000 (50 µg/g), and HSPC (50 µg/g); and (3) cholesterol (100
tions.Amixture of 25 µg/g (cholesterol), 75 µg/g (DSPE-PEG
µg/g), DSPE-PEG 2000 (100 µg/g), and HSPC (100 µg/g).
2000), and 75 µg/g (HSPC) in methanol will serve as QC
12.7 Store the solutions at –20°C.
samples.
12.8 Prepare a solvent blank for a control experiment.
12.2 To prepare ≈ 2.0 g of QC sample, aliquots of 100µL,
300µL, and 300 µL of cholesterol, DSPE-PEG 2000, and
13. Sample Preparation
HSPC, respectively, from individual stock solutions (concen-
tration ≈500 µg/g) is transferred to a 10 mL glass amber vial. 13.1 Two different sample preparation procedures have
To this vial, add ≈1.60 mLof LC/MS-grade methanol and mix beentestedduringthedevelopmentandverificationofthistest
E3323 − 22
method:methanolsolubilization(singlephase)andBligh-Dyer Where w , w , and w indicates mass (in gram) of the empty
1 2 3
extraction (two phase) (21). Methanol solubilization involves tube, mass of the tube + test sample, and mass of tube + test
one-step dilution of the liposomal lipids in methanol, whereas sample + methanol, respectively.
the Bligh-Dyer method involves multiple steps to extract the 13.2.9 Repeat 13.2.4 – 13.2.8 to obtain three independent
methanol solubilized samples and label as 1, 2, and 3.
analytes (lipids) into an organic solvent layer via two-phase
extraction and reconstitution of the analytes in methanol, as 13.2.10 Samples may need to be diluted with LC/MS-grade
methanol appropriately to ensure that the response of the
described in Appendix X3. Unlike methanol solubilization in
which all excipients are dissolved in the solvent, the Bligh- detector is within the calibration range for all analytes. The
dilution factor can be calculated as:
Dyer extraction method minimizes undesirable water-soluble
excipients including salts, sugars, and hydrophilic drug com-
Originalsampleconcentration
Dilutionfactor= (8)
ponents present in liposomal formulations. However, the
Concentrationofdilutedsampleforanalysis
Bligh-Dyer method is a cumbersome and multistep p
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E3323 − 21 E3323 − 22
Standard Test Method for
Lipid Quantitation in Liposomal Formulations Using High
Performance Liquid Chromatography (HPLC) with an
Evaporative Light-Scattering Detector (ELSD)
This standard is issued under the fixed designation E3323; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This test method describes an analytical technique to quantify lipid components that are often present in liposomal
formulations as major components.
1.2 This test method uses high performance liquid chromatography (HPLC) to separate lipids in liposomal formulations and
evaporative light-scattering detection (ELSD) to quantify the individual components.
1.3 This test method quantifies three major organic components in liposomal formulations: cholesterol, 1,2-distearoyl-sn-glycero-
3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000), and hydrogenated soy L-α-
phosphatidylcholine (HSPC).
1.4 This test method can estimate the absolute concentration of cholesterol, DSPE-PEG 2000, and HSPC and their ratio
(DSPE-PEG 2000: HSPC: cholesterol) in liposomal formulations.
1.5 This test method describes preparation of calibration standards and samples, HPLC and ELSD instrumentation, method
development and method validation, sample analysis, and data reporting.
1.6 The detection limits and quantitation limits for the analytes (lipid components) in this test method are in the range of 22 μg ⁄g
to 4 μg/g and 77 μg ⁄g to 10 μg/g, respectively. The analytical measurement ranges for cholesterol, DSPE-PEG 2000, and HSPC
are 1010 μg ⁄g to 165 μg/g, 1010 μg ⁄g to 300 μg/g, and 1010 μg ⁄g to 200 μg/g, respectively.
1.7 Significant digits and rounding of all reported values have been performed according to the guidelines as established in
Practice D6026.
1.8 Units—The values stated in SI units are to be regarded as the standard. Where appropriate, c.g.s units in addition to SI units
are included in this standard.
1.9 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of
regulatory limitations prior to use.
This test method is under the jurisdiction of ASTM Committee E56 on Nanotechnology and is the direct responsibility of Subcommittee E56.08 on Nano-Enabled
Medical Products.
Current edition approved Dec. 15, 2021Oct. 1, 2022. Published April 2022November 2022. Originally approved in 2021. Last previous edition approved in 2021 as
E3323 – 21. DOI: 10.1520/E3323-21.10.1520/E3323-22.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3323 − 22
1.10 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D1193 Specification for Reagent Water
D1356 Terminology Relating to Sampling and Analysis of Atmospheres
D6026 Practice for Using Significant Digits and Data Records in Geotechnical Data
D7439 Test Method for Determination of Elements in Airborne Particulate Matter by Inductively Coupled Plasma–Mass
Spectrometry
E131 Terminology Relating to Molecular Spectroscopy
E177 Practice for Use of the Terms Precision and Bias in ASTM Test Methods
E456 Terminology Relating to Quality and Statistics
E682 Practice for Liquid Chromatography Terms and Relationships
E2490 Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by Photon Correlation Spectroscopy
(PCS)
E3025 Guide for Tiered Approach to Detection and Characterization of Silver Nanomaterials in Textiles
E3080 Practice for Regression Analysis with a Single Predictor Variable
3. Terminology
3.1 Definitions:
3.1.1 accuracy, n—closeness of agreement between a test result and an accepted reference value.
3.1.1.1 Discussion—
The term accuracy, when applied to a set of results, involves a combination of random components and a common systematic error
or bias component. E177
3.1.2 aerosol, n—suspension of solid particles or liquid droplets or both in a gaseous medium. D1356
3.1.3 analyte, n—chemical constituent of interest in an analytical procedure. E3025
3.1.4 analytical instrument qualification, n—collection of documented evidence that an instrument performs suitably for its
intended purpose (1).
3.1.5 baseline noise, n—combination of high-frequency signal fluctuations and low-frequency signal drift that affect baseline
stability.
3.1.5.1 Discussion—
These signal fluctuations can originate from line-voltage fluctuations, shot noise (Poisson noise) from electronic circuits, improper
solvent degassing, temperature instability, and other nonequilibrium effects. Noise is representative of detector response that is not
related to responses from analytes or matrix interferences.
3.1.6 calibration curve, n—relationship between measured response values and analytical concentrations of a standard or reference
material. D7439
3.1.6.1 Discussion—
A set of calibration standards are used to construct a calibration curve, and the concentration of analyte present in an unknown
sample can be determined by comparing the detector response with the calibration curve.
3.1.7 calibration standards, n—set of solutions with known analyte concentration used to construct calibration curves.
3.1.8 carryover effect, n—systematic error that is derived from the preceding sample injection being introduced into the next
sample affecting accurate quantitation.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
The boldface numbers in parentheses refer to a list of references at the end of this standard.
E3323 − 22
3.1.9 cholesterol, n—steroidal organic compound that stabilizes the lipid bilayer in liposomal formulations.
3.1.10 chromatogram, n—graphical presentation of detector response plotted as a function of elution time or effluent volume as
the sample components elute from the column and reach the detector.
3.1.10.1 Discussion—
In the case of evaporative light-scattering detection (ELSD), the detector response is often expressed as voltage (mV) over a range
of elution time (t), where voltage is a function of the intensity of light scattered by nonvolatile particles inside the optical chamber.
For analysis, the characteristic response of ELSD for an eluting analyte is typically evaluated from the peak area under the curve
that is recorded in the chromatogram. This peak area (A) can be expressed mathematically as an integral of detector response for
analytes over an elution time interval from t to t :
1 2
t
A~t! 5* ~S!dt (1)
t
Where A(t) and S(t) = Peak area and the instantaneous detector’s response at time, t, respectively (2).
3.1.11 coeffıcient of determination, n—statistical measure of the linear relationship between X and Y calculated by:
n 2
X Y
S D
( i i
i51
r 5 (2)
n n
X Y
S DS D
( i ( i
i51 i51
Where n = number of observations. E131
3.1.12 evaporation, n—process by which an element or compound transitions from its liquid state to its gaseous state.
3.1.12.1 Discussion—
In the ELSD technique, only the solvent and volatile buffer components of the HPLC-effluent are removed during the evaporation
process and less-volatile or nonvolatile analytes are left behind as dried particles. This process is also called “desolvation”. The
evaporation process depends on various factors including gas pressure, flow rate of the carrier gas, nature of the solvents, and
temperature of the evaporation tube. It is important to choose the appropriate mobile phase components that are volatile;
nonvolatile buffers are not compatible with this test method.
3.1.13 intermediate precision, n—closeness of agreement between test results obtained under specified intermediate precision
conditions. E177
3.1.14 intermediate precision conditions, n—conditions under which test results are obtained with the same test method using test
units taken at random from a single quantity of material that is as nearly homogeneous as possible and with changing conditions
such as operator, measuring equipment, location within the laboratory, and time. E177
3.1.15 limit of detection, n—least amount of analyte in a sample that can be detected but not necessarily quantitated under the
stated experimental conditions.
3.1.15.1 Discussion—
The limit of detection is usually expressed as the concentration of the analyte in the test sample.
3.1.16 limit of quantitation, n—least amount of analyte in a sample that can be quantitatively determined with suitable precision
and accuracy.
3.1.16.1 Discussion—
The limit of quantitation is usually expressed as the concentration of the analyte in the test sample.
3.1.17 linearity, n—ability of the analytical method (within a certain range) to obtain test results that are directly proportional to
the concentration (amount) of the analyte in the sample (3).
3.1.17.1 Discussion—
To establish response linearity, a minimum of six analyte concentrations are recommended. Regression analysis by the method of
least squares (r ) provides a mathematical estimate of the degree of linearity.
E3323 − 22
3.1.18 lipids, n—diverse group of organic compounds that are soluble in organic solvents but are insoluble in water.
3.1.18.1 Discussion—
In this test method, lipids refer to cholesterol, DSPE-PEG 2000, and HSPC. The chemical structures of these three lipids are
presented in Appendix X1.
3.1.19 liposomal formulation, n—product designed to assist in the delivery of an active pharmaceutical ingredient, either
encapsulated or intercalated in a liposome.
3.1.19.1 Discussion—
Formulated products can contain vesicles having a single lipid bilayer (unilamellar), multiple concentric lipid bilayers
(multilamellar), or a mixture of unilamellar and multilamellar vesicles.
3.1.20 liposome, n—synthetic vesicle composed of a one or more bilayers formed by amphipathic molecules such as phospholipids
that enclose a central aqueous compartment. Adapted from (4).
3.1.21 matrix blank, n—substance that closely matches the samples being analyzed with regard to matrix components.
3.1.21.1 Discussion—
Ideally, the matrix blank contains all the sample components except the analyte(s) of interest and is subjected to all
sample-processing operations including all reagents used to analyze the test samples. The matrix blank is used to determine the
presence or absence of any significant interference as a result of the matrix, reagents, and equipment.
3.1.22 matrix effect, n—influence of one or more components from the sample matrix on the measurement of the analyte
concentration or mass.
3.1.22.1 Discussion—
Matrix effects may be observed as increased or decreased detector responses compared with those produced by simple solvent
solutions of the analyte (5).
3.1.23 method validation, n—process used to confirm that an analytical procedure used for a specific test is suitable for its intended
purpose.
3.1.24 mobile phase, n—liquid used to elute sample components through the column that may consist of a single component or
a mixture of components.
3.1.24.1 Discussion—
The term eluent is often used for the preferred mobile phase. E682
3.1.25 nebulization, n—process to convert the solution of target analyte components to a fine-spray via a nebulizer.
3.1.26 peak area, n—area under a peak obtained from integration of a detector signal above the baseline for a given component.
3.1.27 peak resolution, n—measure of chromatographic separation of two components in a mixture calculated by:
t 2 t
~ !
R2 R1
R 5 23 (3)
s
w 1 w
~ !
1 2
where:
R = peak resolution,
s
t and t = retention time of the two components 1 and 2 (t > t ), and
R2 R1 R2 R1
w and w = corresponding widths at the bases of the peaks obtained by extrapolating the relatively straight sides of the peaks
1 2
to the baseline.
3.1.28 precision, n—closeness of agreement between independent test results obtained under stipulated conditions. E177
3.1.29 range, n—interval between the upper and lower concentrations of the analyte in a sample for which it has been
demonstrated that the analytical procedure has an acceptable level of accuracy, precision, and linearity.
E3323 − 22
3.1.30 regression analysis, n—statistical procedure used to characterize the association between two or more numerical variables
for prediction of the response variable from the predictor variable. E3080
3.1.30.1 Discussion—
The objective is to obtain a regression model for use in predicting the value of the response variable for given values of the
predictor variable. In this test method, the response variable is the ELSD signal [light-scattering unit (LSU)] and the predictor
variable is mass concentration.
3.1.31 repeatability, n—precision of test results from tests conducted within the shortest practical time period on identical material
by the same test method in a single laboratory with all known sources of variable conditions controlled at the same levels. Adapted
fromE177
3.1.32 reproducibility, n—precision of test results from tests conducted on identical material by the same test method in different
laboratories. Adapted fromE456
3.1.33 robustness, n—measure of change in the outcome of an analytical procedure with deliberate and systematic variations in
any or all of the key method parameters that influence it. Adapted fromE2490
3.1.34 solvent blank, n—solution containing all reagents used in sample dissolution in the same quantities used for preparation of
blank and sample solutions.
3.1.34.1 Discussion—
The solvent blank is used to assess contamination from the laboratory environment and characterize spectral background from the
reagents used in sample preparation. D7439
3.1.35 specificity, n—ability to assess unequivocally the analyte in the presence of components that may be expected to be present
in the test sample.
3.1.35.1 Discussion—
Typically, these might include impurities, degradants, matrix, and so forth (3).
3.1.36 system suitability, n—determination of instrument performance in a particular procedure (for example, sensitivity and
chromatographic retention) by analyzing a set of appropriate reference standards before the analytical run.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 test sample, n—final form of the sample that is used for testing.
3.2.1.1 Discussion—
In this test method, the sample solubilized in methanol followed by appropriate dilution by solvent is defined as the test sample.
3.2.2 test unit, n—unit or portion of a material that is obtained from a primary material following a sampling procedure to acquire
test result(s) for the property(-ies) to be measured.
3.2.2.1 Discussion—
In this test method, the original liposomal formulation to be tested for lipid quantitation is defined as the test unit.
3.3 Acronyms:
3.3.1 Cal—Calibration
3.3.2 CRM—Certified reference material
3.3.3 DSPE-PEG—1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)
3.3.4 ELSD—Evaporative light-scattering detector
3.3.5 HPLC—High performance liquid chromatography
3.3.6 HSPC—Hydrogenated soy L-α-phosphatidylcholine
E3323 − 22
3.3.7 ID—Inside diameter
3.3.8 LOD—Limit of detection
3.3.9 LOQ—Limit of quantitation
3.3.10 LSU—Light-scattering unit
3.3.11 OSHA—Occupational Safety and Health Administration
3.3.12 QA—Quality assurance
3.3.13 QC—Quality control
3.3.14 RCF—Relative centrifugal force
3.3.15 RSD—Relative standard deviation
3.3.16 SD—Standard deviation
3.3.17 SLM—Standard liter per minute
3.3.18 ULOQ—Upper limit of quantitation
3.3.19 UV—Ultraviolet
4. Summary of Test Method
4.1 Over the past few decades, several liposomal drug formulations have been approved for clinical use (6, 7). An ongoing effort
from pharmaceutical industries, academic institutions, foundations, and industry partners has been made to develop new nanoscale
liposomal formulations with improved drug efficacy, and many products are currently being assessed in clinical trials (7-11). The
critical quality attributes to consider for these nanomaterials include size and shape heterogeneity, chemical composition, and
physicochemical stability of ingredients present in the liposome formulation (12).
4.2 This test method describes an analytical method for the separation and quantitation of cholesterol, DSPE-PEG 2000, and
HSPC in liposomal formulations.
4.3 This test method is based on the combination of two well-established analytical techniques: (1) chromatographic separation
of analytes (a mixture of cholesterol, DSPE-PEG 2000, and HSPC) via HPLC using a designated column, and (2) quantitation of
analytes present in the effluent via ELSD in which the scattering intensity of the incident light is correlated with the total mass of
the analytes present in the injected sample. The ELSD detector can serve as a universal detector for a range of nonvolatile or
semi-volatile analytes including those that do not have ultraviolet (UV)-absorbing chromophores (13, 14).
4.4 A calibration curve from six calibration standards of cholesterol, DSPE-PEG 2000, and HSPC is developed by following the
procedure described in this test method. As ELSD shows a nonlinear response with analyte concentration, logarithmic
transformation is performed to obtain a linear regression model. Hence, log (peak area) versus log (concentration) is plotted for
each analyte to obtain the corresponding calibration curve. The slope and intercept obtained from linear regression analysis are
used to quantify the individual mass (concentration) of lipid components in an unknown liposome sample.
4.5 This test method describes the specific test conditions, sample preparation, method validation, and data analysis requirements.
E3323 − 22
5. Significance and Use
5.1 Lipid composition in a liposomal formulation is an important aspect during synthesis of liposomes, which determines stability,
surface characteristics, drug encapsulation, and drug release capabilities. The cholesterol component plays a key role in controlled
drug release by adding stability to the liposome. A small variation in the lipid composition can significantly alter the parameters
mentioned above (15).
5.2 Variation in the lipid composition in the liposomal formulation may influence the safety and efficacy of the product. Therefore,
chemical composition of the liposomes shall be determined.
5.3 The pharmaceutical industry and regulatory agencies require QC, QA, specifications, thorough characterization, and
quantification of lipid components (16, 17).
5.4 This test method can be used to ascertain variations in the lipid component profiling of various liposomal formulations.
However, this test method does not intend to identify chemical degradation products (18).
5.5 Analyzing the stability of analytes and their chemical degradation profiles as a result of oxidation or hydrolysis is beyond the
scope of this test method (18, 19).
6. Interferences
6.1 Method interferences may be introduced by impurities present in reagents, glassware, and other apparatus used during sample
preparation and instrumental analysis. These impurities may result in high baseline noise or interfering peaks. The presence and
magnitude of method interferences are determined by routine analysis of solvent and laboratory blanks.
6.2 To avoid heterogeneity in pH of the solution, eluents and buffer salts shall be properly mixed (sonication is recommended).
It is also recommended that the solvent reservoir bottle be cleaned routinely, and the appropriate bottles be filled with freshly
prepared mobile phase solutions.
6.3 Aerosol formation in ELSD requires a constant supply of dry and filtered gas that is free from particulate matter and
nonvolatile hydrocarbons. The most commonly used gas is nitrogen. The inlet gas shall be filtered through a 0.01 μm filter to
remove particulate matter, and an appropriate gas adsorbent trap should be used to remove nonvolatile hydrocarbons and moisture,
and thereby minimizing baseline noise. The use of gases that allows either combustion of solvents or oxidation of target analytes
should be avoided.
6.4 Keep the autosampler injection port and column clean to avoid carryover or ghost peaks. Contamination of glass containers
or vials used for this test method should be avoided.
6.5 Fluctuations in detector response can adversely affect data quality. To avoid this issue, stabilize the detector at the experimental
conditions (for example, nebulization temperature, evaporation temperature, and nitrogen flow rate) for 30 min. This will also
minimize baseline noise.
6.6 Set the temperature of the column compartment as recommended in the test method and equilibrate the column while
stabilizing the detector response (see 6.5).
6.7 The presence of bubbles in the HPLC tubing causes pressure fluctuations. Solvent purging (2.00 mL/min) for 15 min in which
the column is bypassed is recommended to remove interferences caused by the presence of air bubbles.
6.8 Excipients present in the test samples may interfere with the detection of cholesterol, DSPE-PEG 2000, or HSPC. Therefore,
the matrix effect can be assessed by comparing the detector response for a known amount of standard analyte spiked in the matrix
blank and for the same amount of analyte spiked in a solvent blank. If any matrix effect is observed by this comparison,
optimization of the sample preparation procedure to remove the interfering compounds from the test sample or modification of the
chromatographic parameters (for example, solvent gradient or eluent flow rate) to avoid the coelution of target analytes and
E3323 − 22
interfering excipients are recommended. The Bligh-Dyer method, as described in Appendix X3, could be adopted to remove
water-soluble excipients in test liposomal formulations, if necessary.
6.9 Chemicals with high purity shall be used for the preparation of lipid calibration standards. When feasible, it is recommended
that higher-order reference standards (for example, CRMs) are used in calibration. If reference materials are not available,
high-quality crystalline or lyophilized chemicals of known purity can be used for this purpose.
6.10 All the stock solutions, calibration standards (calibration levels), and test samples should be stored either at 00 °C to 4 °C
or –20°C–20 °C as recommended in this test method to avoid degradation of target analytes.
7. Apparatus
7.1 HPLC, with in-line degasser module.
7.2 ELSD.
7.3 Deactivated BEH (bridged ethylene hybrid) C18 column, with 13 nm pore size, 3.5 μm particle size, and 3 mm ID × 150 mm
column length or other equivalent stable C18 column that can resolve the peaks for analytes and potential interference with a peak
resolution ≥1.5.
7.4 Analytical balance that can accurately weigh with ≤0.0001 g readability.
7.5 Vortex mixer.
7.6 Mechanical pipettors, covering ranges from 22 μL to 20, 10020 μL, 100 μL to 200,200 μL, and 100100 μL to 1000 μL and 10
mL.
7.7 Glass amber vials, 1010 mL and 20 mL.
7.8 Autosampler amber vials, 2 mL.
7.9 Solvent reservoir bottle, 1 L.
7.10 Bottle top vacuum filter system, pore size 0.2 μm.
7.11 Ultrasonic water bath.
8. Reagents and Materials
8.1 Purity of Reagents—Reagent-grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society where such
specifications are available. Other grades may be used, provided it is first ascertained that the reagents are of sufficiently high
purity to permit their use without lessening the accuracy of the determinations.
8.2 Reagents—All reagents should be of LC-MS grade and solvents should be prefiltered with a ≤0.2 μm filter. Other grades (for
example, HPLC grade) may be used, provided it is first ascertained that the reagents are of sufficiently high purity to permit their
use without lessening the accuracy of the determinations.
8.2.1 Ammonium acetate, LC-MS grade.
ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference Materials, American Chemical Society, Washington, DC. For
suggestions on the testing of reagents not listed by the American Chemical Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and
the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.
E3323 − 22
8.2.2 Acetonitrile, LC-MS grade.
8.2.3 Methanol, LC-MS grade.
8.2.4 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean reagent water as defined by
Type 1 of Specification D1193. Use deionized water (>18 MΩ cm) Type 1 high-purity water.
8.3 Materials:
8.3.1 Lipid Standards:
8.3.1.1 Cholesterol, ≥99 % pure, powder form.
8.3.1.2 HSPC, ≥99 % pure, consisting of C16:0 (HSPC 1) and C18:0 (HSPC 2) fatty acids, powder form.
8.3.1.3 DSPE-PEG 2000, ≥99 % pure, powder form.
9. Hazards
9.1 Because the ELSD produces an aerosol, the outlet of the ELSD unit shall be vented to a suitable fume hood or external exhaust.
9.2 Follow laboratory safety protocol and proper protective measures while handling liposomal formulations.
9.3 This test method uses methanol, chloroform, and acetonitrile that exhibit various levels of toxicity through inhalation and skin
contact. All organic solvents used in this test method should be handled in a chemical fume hood. Avoid inhalation of the solvents.
All the organic wastes should be disposed of appropriately.
9.4 The waste of the sample extract should be handled with proper care and precaution. Follow laboratory safety protocol to
appropriately dispose of used test sample. The user is advised to follow relevant local regulatory requirements (for example,
OSHA), suppliers’ safety data sheets, institutional requirements, and recommended procedures pertaining to safe handling and
disposal of all chemicals used in this test method.
10. Preparation of Mobile Phases
10.1 Mobile Phase A: Acetonitrile/Water (90/10 v/v) + 5 mmol ⁄L Ammonium Acetate:
10.1.1 Filter deionized water (>18 MΩ cm) through a 0.2 μm membrane using a bottle top vacuum filter system. This step is not
needed in the case of a water purification system with an attached 0.2 μm filter.
10.1.2 Rinse an empty solvent bottle (1 L) with deionized water thoroughly and dry the bottle.
10.1.3 In a 1 L volumetric flask, transfer 3.853.85 g 6 0.02 g of ammonium acetate quantitatively, dissolve the salt thoroughly
with ≈800 mL of deionized water, equilibrate the solution at room temperature, and then fill the flask with deionized water to the
1 L graduation mark. This will provide 50 mmol ⁄L solution of ammonium acetate.
10.1.4 Transfer 100 mL of 50 mmol/L ammonium acetate and 900 mL of LC-MS-grade acetonitrile in the clean and dry 1 L bottle.
The volumes are measured using a graduated cylinder (100 mL and 1 L, respectively).
10.1.5 Degas the solution for 1010 min to 15 min using an ultrasonic bath before using it as mobile phase A. The use of an in-line
degasser module further helps to achieve a stable baseline during an analytical run.
10.1.6 Transfer the degassed solution (mobile Phase A) to an empty, clean, and dry 1 L reservoir attached to the HPLC.
10.2 Mobile Phase B: Methanol + 5 mmol/L Ammonium Acetate:
10.2.1 To prepare 1 L of mobile Phase B, weigh 385385 mg 6 10 mg of ammonium acetate and transfer it quantitatively to an
E3323 − 22
empty 1 L volumetric flask, fill the flask with ≈800 mL of LC-MS-grade methanol to dissolve ammonium acetate, equilibrate the
solution at room temperature, and then fill the flask with methanol to the 1 L graduation mark to make a homogeneous 5 mmol/L
ammonium acetate solution in methanol.
10.2.2 Transfer the methanolic 5 mmol/L ammonium acetate solution to a clean and dry 1 L bottle.
10.2.3 Sonicate the solution to homogenize the analytes as needed and degas the solution for 1010 min to 15 min using an
ultrasonic bath. Use of an in-line degasser module further helps to achieve a stable baseline during an analytical run.
10.2.4 Transfer the degassed methanol with 5 mmol/L ammonium acetate to an empty, clean, and dry 1 L reservoir on HPLC.
NOTE 1—Sparging with helium can be used as an alternative to the ultrasonic degassing and in-line vacuum degassing combination as recommended in
this test method.
11. Preparation of Calibration Standards
NOTE 2—Gravimetric Measurements—All working solutions in this test method are prepared gravimetrically using an analytical balance (0.0001 g
accuracy). The concentration of an analyte is expressed in units of μg analyte per g of solution. Although the volumetric preparation shows close
agreement with the gravimetric preparation, it is known that a 1 % to 5 % error can be introduced during small volume transfers and, hence, bias the
quantitation results. The analytical balance provides better measurement resolution (that is, more significant figures) than mechanical pipettes and offers
better accuracy.
NOTE 3—Conditioning the pipette tip with appropriate solvents before transfer of calibration standards for weighing, working promptly with the stock
solutions, and weighing the volatile liquids in securely capped containers using a secondary container are highly recommended practices.
NOTE 4—The user is required to provide a certificate of analysis or equivalent alternative information on the source, purity, storage conditions,
retest/expiration date, and lot/batch number of reference standards or high-purity chemicals to ensure quality and stability (20).
11.1 Powder stocks of the analytes stored in the freezer at –20 ºC should be allowed to equilibrate at ambient temperature before
weighing.
11.2 Deionized water (>18 MΩ cm) should be filtered through a 0.2 μm membrane using a bottle top vacuum filter system before
use. This step is not needed in the case of water purification system with an attached 0.2 μm filter.
11.3 Preparation of Individual Stock Solutions—Prepare individual stock solutions (≈1000 μg/g) of cholesterol, DSPE-PEG 2000,
and HSPC in separate amber glass vials (20 mL).
11.3.1 Rinse three empty 20 mL amber glass vials with deionized water and dry thoroughly.
11.3.2 Label each vial with the corresponding analyte j, where j = cholesterol, DSPE-PEG 2000, and HSPC. Put a cap on each
bottle.
11.3.3 Weigh each capped vial on an analytical balance and record the mass to 60.1 mg as W .
0j
11.3.4 Individually weigh 1010 mg 6 2 mg of cholesterol, DSPE-PEG 2000, and HSPC on an analytical balance, transfer each
analyte to the appropriately labeled vial, put a cap on each vial, and record the mass of the capped vial + analyte as W .
1j
11.3.5 Transfer 12.5 mL (≈10.0 g) of LC-MS-grade methanol using a mechanical pipettor to the vial from 11.3.2 labeled as
cholesterol. Put a cap on each vial.
11.3.6 Repeat 11.3.5 for DSPE-PEG 2000 and HSPC.
11.3.7 Weigh each capped vial containing, analyte j, and methanol on an analytical balance and record the mass to 62 mg as W .
2j
11.3.8 Dissolve the solids in each vial thoroughly by vortex mixing for 1 min.
NOTE 5—Complete solubilization and formation of a homogeneous solution may require an additional 5 min bath sonication.
E3323 − 22
11.3.9 Store the vials of the individual stock solutions at –20 °C until needed. Stock solutions are stable up to four months under
this condition.
11.3.10 The individual stock concentrations of the three components, C , are calculated using Eq 4. The purity (j) % for each
j
component should be the value from the manufacturer’s certificate of analysis:
Mass of the analyte
C 5 5
j
Total mass of solution
~purity ~j!%!3@~W 2 W ! ⁄ ~W 2 W !# 310 µg/g (4)
1j 0j 2j 0j
Where j = cholesterol, DSPE-PEG 2000, or HSPC.
11.4 Preparation of Individual Stock Solutions—Prepare individual stock solutions (≈500 μg/g) of cholesterol, DSPE-PEG 2000,
and HSPC in separate amber glass vials (20 mL).
11.4.1 Rinse three empty 20 mL glass vials with deionized water and dry thoroughly.
11.4.2 Label each vial with the corresponding component (analyte) j, where j = cholesterol, DSPE-PEG 2000, or HSPC. Put a cap
on each vial.
11.4.3 Follow 11.3.3 – 11.3.8 (with a lesser amount of analyte j, for example, 55 mg 6 1 mg) to obtain Cj values of 500 μg ⁄g.
11.4.4 The concentration of each analyte j in the individual stock solutions is calculated using Eq 4.
11.4.5 Store the vials of the individual stock solutions at –20 °C until needed. Stock solutions stored under this condition are stable
up to four months.
11.5 Preparation of Calibration Standards for Individual Analyte:
11.5.1 Six calibration standards (that is, calibration levels) of individual analytes: (1) cholesterol with target concentrations
ranging from 55 μg ⁄g to 175.0175 μg/g (for example, 5.0,5 μg 10.0,⁄g, 10 μg 25.0,⁄g, 25 μg 50.0,⁄g, 50 μg 100.0,⁄g, 100 μg ⁄g, and
175.0175 μg/g); (2) DSPE-PEG 2000 with target concentrations ranging from 5.05 μg ⁄g to 300.0300 μg/g (for example, 5.0,5 μg
10.0,⁄g, 10 μg 50.0,⁄g, 50 μg 100.0,⁄g, 100 μg 150.0,⁄g, 150 μg ⁄g, and 300.0300 μg/g); and (3) HSPC with target concentrations
ranging from 5.05 μg ⁄g to 200.0200 μg/g (for example, 5.0,5 μg 10.0,⁄g, 10 μg 50.0,⁄g, 50 μg 100.0,⁄g, 100 μg 150.0,⁄g, 150 μg ⁄g,
and 200.0200 μg/g) are prepared from the 1000 μg/g and 500 μg/g stock solutions (see 11.3 and 11.4 respectively). ELSD has
stronger analytical sensitivity for cholesterol compared to DSPE-PEG 2000 and HSPC, therefore the highest calibration level for
cholesterol is targeted at ≈175.0≈175 μg/g. In this test method, all measurements are gravimetric, and therefore, the user does not
need to transfer the exact volume of methanol or analyte stock solution to reach the exact target analyte concentration but does
need to record the accurate mass and calculate the concentration of each calibration standard by following the example described
in 11.5.7 – 11.5.9.
11.5.2 Rinse one empty 10 mL glass vial with deionized water and dry thoroughly.
11.5.3 Rinse one measuring cylinder with deionized water and dry thoroughly.
cal
11.5.4 For calibration Level 1, C = 5 μg/g, where j = cholesterol, DSPE-PEG 2000, or HSPC. Label the 10 mL vial as Level
j
1 (cholesterol, DSPE-PEG 2000 or HSPC). Put a cap on the vial.
11.5.5 For that level, weigh the capped vial on an analytical balance and record the mass to 60.1 mg as W .
0j
11.5.6 Add the volume of the stock solution with analyte j = cholesterol to the vial as given in Table 1 for Level 1. Place a cap
on the vial. Record the mass as A in units of g.
1j
11.5.7 Add the volume of methanol to the vial as given in Table 1 for Level 1. Place a cap on the vial. Record the mass as A
2j
in units of g. The three masses, W , A , and A , will be required to calculate the actual mass of analyte j present in the solution
0j 1j 2j
and the total mass of analyte j plus solvent (that is, methanolic solution of analyte j).
E3323 − 22
A
TABLE 1 Example of Six Calibration Standards for Individual Analyte Prepared from Two Stock Solutions (1000 and 500 μg/g)
Amount of analyte needed (C )
Target conc. of analyte Conc. of stock used Total mass of the j Amount of methanol
Cal. level
in methanol (μg/g) (ppm, μg/g) = C target cal. standard (g) added (mL)
j Mass (μg) Stock Volume (mL)
1 5 500 4 20 0.05 2.43
2 10 500 4 40 0.10 2.38
3 50 500 4 200 0.25 2.30
4 100 500 4 400 0.50 2.15
5 150 1000 4 600 0.76 1.77
6 300 1000 4 900 1.52 1.39
A
TABLE 1 Example of Six Calibration Standards for Individual Analyte Prepared from Two Stock Solutions (1000 and 500 μg/g)
Amount of analyte needed (C )
Target conc. of analyte Conc. of stock used Total mass of the j Amount of methanol
Cal. level
in methanol (μg/g) (ppm, μg/g) = C target cal. standard (g) added (mL)
j Mass (μg) Stock Volume (mL)
1 5 500 4 20 0.05 5.00
2 10 500 4 40 0.10 4.95
3 50 500 4 200 0.25 4.80
4 100 500 4 400 0.50 4.55
5 150 1000 4 600 0.76 4.29
6 300 1000 4 1200 1.52 3.35
A
To estimate the volume of analyte stock solution, the density of methanol is used as the density of stock. Other calibration standards with intermediate analyte
concentration, for example, concentrations of 25,25 μg 75,⁄g, 75 μg 175,⁄g, 175 μg ⁄g, or 200 μg/g can be prepared similarly.
11.5.8 The concentrations of six stock solutions for three analytes, C (μg/g) (≈1000 μg/g and ≈500 μg/g) are precalculated in
j
11.3.10 and 11.4.5, respectively. These values are used in 11.5.9 to calculate the concentration of a calibration standard.
11.5.9 The final concentration of the calibration standard for analyte j is determined by Eq 5:
cal
C µg/g 5 C 3@ A 2 A ⁄ A 2 W # (5)
~ ! ~ ! ~ !
j j 2j 1j 1j 0j
11.5.10 Repeat 11.5.2 – 11.5.9 for calibration levels 2 to 6, using volumes of each analyte stock solution and methanol shown in
Table 1.
11.5.11 Repeat 11.5.2 – 11.5.10 for the other two analytes, j = DSPE-PEG 2000 and HSPC.
11.5.12 Homogenize each individual analyte + methanol solution for calibration levels 1 to 6 by vortex mixing. Store the
calibration solutions at 00 °C to 4 °C (for one day) or –20 °C for longer storage. The solutions are stable up to one month at –20 °C.
11.5.13 A representative table for the six calibration standards with the target analyte concentrations is provided in Table 1.
NOTE 6—The concentrations provided in Table 1 are the examples of target concentrations for various calibration levels. Eq 5 shall be used to obtain
mass-based concentrations for any target analyte concentration.
11.5.14 The individual calibration plots are used only to verify the calibration range in which the regression model and the fit
remains linear, and to choose the concentration range for preparing calibration standards containing mixture of the three analytes.
11.6 Preparation of Calibration Standards of Analyte Mixtures:
11.6.1 Mixtures of cholesterol, DSPE-PEG 2000, and HSPC are utilized to construct calibration curves containing a minimum of
six calibration levels, and the constructed calibration curve within the bracketed range is used for method validation and the
quantitation of all three analytes present in the test samples.
11.6.2 Each calibration standard (level) is obtained by combining the three stock solutions of individual analytes (either
≈1000≈1000 μg ⁄g or ≈500 μg/g stock) in a 1:1:1 mass ratio with methanol in a 10 mL vial. Example of solution preparation
parameters for various concentration levels (mass ratio of cholesterol: DSPE-PEG 2000: HSPC ≈ 1:1:1) are summarized in Table
2.
11.6.3 To prepare the eight levels of the calibration standard mixtures shown in Table 2, follow the steps similar to those described
in 11.5.2 – 11.5.7.
E3323 − 22
A
TABLE 2 Example of Calibration Standards of Analyte Mixtures Prepared from Two Stock Solutions (1000 and 500 μg/g)
Target conc. of each
Amount of analyte needed (C )
j
Conc. of stock used Total mass of the Amount of methanol
Cal. level analyte in the mix
(ppm, μg/g) = C target cal. standard (g) added (mL)
j
Mass (μg) Stock Volume (μL)
(μg/g)
1 5 500 2 10 25.0 2.43
2 10 500 2 20 50.0 2.38
3 15 500 2 30 75.0 2.30
4 25 500 2 50 125.0 2.15
5 50 1000 2 100 125.0 2.15
6 75 1000 2 150 188.0 1.95
7 200 1000 2 300 500.0 1.00
8 300 1000 2 600 750.0 0.25
A
TABLE 2 Example of Calibration Standards of Analyte Mixtures Prepared from Two Stock Solutions (1000 and 500 μg/g)
Target conc. of each
Amount of analyte needed (C )
j
Conc. of stock used Total mass of the Amount of methanol
Cal. level analyte in the mix
(ppm, μg/g) = C target cal. standard (g) added (mL)
j
Mass (μg) Stock Volume (μL)
(μg/g)
1 5 500 2 10 25 2.43
2 10 500 2 20 50 2.38
3 15 500 2 30 75 2.30
4 25 500 2 50 125 2.15
5 50 1000 2 100 125 2.15
6 75 1000 2 150 188 1.95
7 200 1000 2 400 500 1.00
8 300 1000 2 600 750 0.25
A
To estimate the volume of analyte stock solution, the density of methanol is used as the density of stock. The analyst is not limited to example concentrations, and any
six concentrations (evenly spaced) can be used to establish a linear fit with r $ 0.995. Other calibration standards with intermediate analyte concentration, can be prepared
similarly.
11.6.4 Rinse the eight empty 10 mL glass vials with deionized water and dry thoroughly.
11.6.5 Label each vial with the corresponding calibration level (1 to 8).
11.6.6 Add appropriate volumes of the three individual stock solutions (cholesterol, DSPE-PEG 2000, and HSPC) and methanol
as given in Table 2 for the calibration level. Place a cap on each vial.
11.6.7 Record the mass of each capped vial after the addition of each analyte solution.
11.6.8 Add the appropriate volume of methanol to each vial as given in Table 2 for the calibration level. Place a cap on each vial.
11.6.9 Record the mass of each capped vial after the addition of methanol.
11.6.10 The final concentration of an individual analytes j (where j = cholesterol, DSPE-PEG 2000, or HSPC) in a calibration
standard mixture can be calculated as:
cal
C ~µg/g!
j
5 mass of analyte added, µg ⁄ total mass of analyte 1 methanol, g (6)
@~ !~ !#
cal
C µg/g 5
~ !
j
(6)
mass of analyte added, μ g ⁄ total mass of analyte+methanol, g
@~ ! ~ !#
Where the mass of analyte j added (μg) = C × (mass of the individual stock solution added, g).
j
11.6.11 Homogenize the mixture by vortex mixing the solution. Store these calibration standard mixtures at 00 °C to 4 °C (for one
day) or –20 °C for longer storage. Stock solutions are stable up to one month at –20°C.–20 °C.
12. Preparation of Samples for QC, Method Specificity, Recovery Experiment, and Method Precision
12.1 QC samples are to be prepared by mixing cholesterol, DSPE-PEG 2000, and HSPC from the 500 μg/g stock solutions. A
mixture of 25 μg/g (cholesterol), 75 μg/g (DSPE-PEG 2000), and 75 μg/g (HSPC) in methanol will serve as QC samples.
12.2 To prepare ≈ 2.0 g of QC sample, aliquots of 100, 300,100 μL, 300 μL, and 300 μL of cholesterol, DSPE-PEG 2000, and
HSPC, respectively, from individual stock solutions (concentration ≈500 μg/g) is transferred to a 10 mL glass amber vial. To this
E3323 − 22
vial, add ≈1.60 mL of LC/MS-grade methanol and mix thoroughly using vortex mixer. Masses in each step shall be recorded. The
component concentrations in the QC sample are determined based on the masses of each of three analytes and the mass of methanol
solvent that have been added.
12.3 To assess the specificity: (1) prepare a mixture of cholesterol, DSPE-PEG 2000, and HSPC having each analyte concentration
of 50 μg/g in methanol as described in Table 2; (2) prepare a matrix blank which is a methanol solution containing possible matrices
that are expected to be present in the test sample (that is, liposomal formulation); (3) prepare a solvent blank containing only
methanol; and (4) prepare three independent solutions of cholesterol (50 μg/g), DSPE-PEG (50 μg ⁄g), and HSPC (50 μg ⁄g) in
methanol as described in Table 1.
12.4 For the accuracy study, prepare three replicates in methanol of a set of samples with the following analyte concentrations:
(1) (low) mixture of cholesterol, DSPE-PEG 2000, and HSPC with each analyte concentration of 25 μg/g; (2) (medium) mixture
of cholesterol, DSPE-PEG 2000, and HSPC with each analyte concentration of 50 μg/g; and (3) (high) mixture of cholesterol,
...